The endoplasmic reticulum (ER) is the site of synthesis of virtually all secreted proteins. Hydrophobic signal sequences at the amino terminal of secretory proteins are the key to their entry to the ER. An increasing body of evidence demonstrates that specific proteins in the cytosol and the ER membrane bind to signal sequences during the process of protein translocation across the ER membrane. Removal of the signal sequence from the precursor protein by the action of signal peptidase release the mature protein to the ER lumen. The fate of the cleaved signal peptide is poorly understood, but since the signal peptide is believed to bind to components of the translocation apparatus, its removal and or degradation may be important in maintaining the availability of the translocation apparatus for continued rounds of protein transport. It is now apparent that at least some signal peptides undergo a specific proteolytic processing event by a microsomal signal peptide peptidase (MSPP). Preliminary data suggest that MSPP may be related to an analogous peptide from E. coli, protease IV. The long-term objectives of this proposal are to purify MSPP from mammalian rough microsomes and to establish a cell-free system in the yeast, Saccharomyces cerevesiae, to enable future genetic studies on the role of MSPP. Using solubilized microsomal membranes, the following specific aims are proposed: (1) Characterize the solubility of mammalian MSPP and optimize direct and indirect assays for its activity. (2) Purify MSPP from mammalian microsomes. (3) Establish cell-free assays for MSPP activity in the yeast, Saccharomyces cerevisiae. MSPP is likely to be an important component of the translocation apparatus and may be involved in other cellular proteolytic processes as well. These studies are essential for elucidating the role of this newly described enzyme in eukaryotic cell biology. An increasing number of human diseases characterized by deficiencies in specific secretory proteins (e.g. hormones, clotting factors) have been ascribed to defects in signal sequence function in the deficient protein. In some cases the uncleaved proteins may accumulate in the cell in which they are synthesized, and may result in disrupted cell function as well as insufficient secretion of the specific protein. Thus, an understanding of normal signal sequence fat and function is important not only for our fundamental understanding off secretory protein biogenesis, but for understanding the full medical implications of abnormal signal sequence function. Moreover, an understanding of basic differences between eukaryotic and bacterial secretory machinery will identify targets for the future development of novel antibiotics.